PCR has become a rapid and reliable tool for the molecular biology-based diagnosis of a variety of infectious diseases from a wide range of clinical samples. Fecal specimens, the sample type used in our GPP assay, are among the most complex specimens for PCR. Therefore, it must be emphasized that for such a sensitive and complex assay, every element of PCR, no matter how simple or mundane, can affect the outcome of results. This increased sensitivity is critical for low copy pathogen detection and the amplification of degraded samples, as well as for multiplex PCR.
What happens prior to thermal cycling is critical to the success of PCR. We recommend paying particular attention to the temperature of the reagents during PCR set up because the Taq DNA polymerase retains some activity, even at room temperature.
Thus, under non-stringent annealing conditions, such as at room temperature, products can be generated from annealing of primers to target DNA at locations of low complementarity or having complementarity of just a few nucleotides at the 3′end. This can, in effect, create new templates attached with the primer sequences. Ensuing cycles amplify these attached sequences, generating nonspecific products and possibly reducing amplification efficiency of specific products by competition for substrates or polymerase.
Fortunately, focusing on keeping your reactions cold during PCR setup, preheating the thermocycler, and preheating the plate on the Luminex instrument will help you obtain consistently high-quality data and ensure the integrity and economic use of your GPP reagents. Remember, when it comes to PCR, shortcuts backfire more often than not.
The following problems can be a result of temperature-related issues:
- Consistently high background for a problematic target
- Random non-specific signal across several targets
- High background for most or all targets
- Low signal
To make your life easier, make sure to focus on the following points:
- Prior to beginning the assay, go through the GPP product insert and highlight the pre-heating reminders so you don’t overlook them.
- Keep everything cold! — Keep PCR reagents cold throughout Master Mix setup and Sample addition.
- Place PCR reagents in a cold block after thawing.
- Keep the Master Mix on ice or in a cold block.
- Use a PCR cooler rack (96-well cold block) to keep reaction aliquots cold during sample addition.
- Prepare Master Mix and add samples in a timely fashion, but don’t compromise proper technique or thorough mixing to save time.
- Pre-heat the thermal cycler to 53°C prior to RT-PCR.
- Quickly transfer the tubes from the cold block to the preheated thermocycler.
- Run in Block mode (BioRad and Eppendorf) with heated lid enabled.
- 53°C incubation temperature provides highly specific reverse transcription conditions resulting in lower background and increased yield of specific product.
- Pre-heat thermal cycler to 60o C prior to Bead Hybridization to avoid high background.
- Perform Hybridization/Detection step within 12 hours of PCR completion.
- Pre-heat the Luminex XYP heater block to 45°C. Keeping the reactions at 45°C after the Bead Hybridization step until the time of data acquisition is essential for reduced background signals.
- The temperature of the Luminex XYP heater block should be set to 45°C at least 10 minutes before the completion of the hybridization/detection.
- The hybridization reaction should be transferred to the heated Luminex XYP block after the completion of the 45o C – 45 minute step. The additional 45o C – Hold step is to prevent the hybridization reaction from cooling if not immediately removed from the thermocycler.
- Efficiently transfer the hybridization reaction from the thermocycler to the Luminex to prevent cooling.